1. Field of the Invention
The present invention relates to cell lines established from insect tissues, especially relates to cell lines from Maruca vitrata with high susceptibility to insect pathogenic microorganisms such as Maruca vitrata multiple nucleopolyhedrovirus (MaviMNPV), which can be used in the mass production of MaviMNPV and recombinant proteins.
2. The Prior Arts
Maruca vitrata belongs to Order of Lepidoptera, and Family of Pyralidae. Host plants include 5 families: the legume family, the Pedaliaceae, the Mimosaceae, the Caesalpiniaceae, and the Malvaceae, total of 20 genera and 40 crops. It spreads widely in Asia, Africa, and the Pacific islands, and is found in fields of Taiwan all year-round. Larvae of M. vitrata feed on leaflets and buds, bore into floral buds and pods, and spin the leaves, blossoms and pods together to conceal themselves inside. Control of M. vitrata by administration of pesticides is difficult since the larvae hide inside the plants. Therefore, the quality and yields of the crops are dramatically affected. In southern area of Taiwan, the yield losses of the legume family due to M. vitrata can be as high as 30%. Recently the government promotes the use of green manure legumes. Sesbania cannabina, Crotalaria junce, and soybean are served as green manure during fallow to improve soil fertility, which are happened to be the host plants for M. vitrata. This strategy increased planting areas of the green manure legumes consistently. In addition, farmers seldom use pesticides during planting these green manures. The green manure legumes hence become the food supply for M. vitrata, and the population densities of M. vitrata significantly increased to cause the damage of legumes. On the other hand, M. vitrata is an important quarantine pest to yard long bean in Africa, which is also the main factor for harvest loss in yard long bean. Up to 80% yield losses of yard long bean due to M. vitrata has often been observed in West Kenya. Therefore the damage of M. vitrata to economical plants and the effective control of M. vitrata have become important issues internationally.
The chemical insecticides are commonly used to kill the larvae of M. vitrata to control the spread of M. vitrata. However, chemical control is complicated by the fact that larvae spin silken threads and hide inside, which also causes the problems of insecticide resistance and residual effects, or involved in environmental contamination and resurgence of secondary insect pest species. Therefore, alternative control methods are studied to replace or to reduce the dependence of chemicals. Among them, the bioinsecticide based on baculovirus production is the most important control method. Studies have revealed that M. vitrata multiple nucleopolyhedrovirus (MaviMNPV) is the pathogen for nucleopolyhedrosis and the cause of death for M. vitrata larvae. MaviMNPV belongs to the baculovirus family, which generates budded virus (BV) after infection. The occlusion bodies are released from the membrane of host cells by budding, spread infection between cells inside the insect body, and start the polyhedrin gene to express polyhedron. Multiple virions are found embedded in polyhedrin protein in the nucleus known as an occlusion body (OB), which is also referred to as a polyhedron inclusion body (PIB), and the virus is therefore named as multiple nucleopolyhedrovirus (MNPV). The occlusion bodies can be released outside from the lysed infected insects and further infect other insects to induce an epizootic disease. The NPVs are highly host-specific and infects only invertebrates but not humans, animals or other organisms. The spread of the NPV is not only caused by horizontal transmission from insects to insects or ingestion of the occlusion bodies, but also be spread through vertical transmission from generations to generations. Studies have shown that the MaviMNPV virus-induced mortality can be up to 98% in second instar larvae of M. vitrata. Therefore the MaviMNPV can be used as safe biological control agents in prevention of pests and M. vitrata. In addition, MaviMNPV belongs to the baculovirus family; the latter is a core expression system of foreign protein production for medical and industrial applications. This indicates the potential of being a recombinant protein expression system for MaviMNPV.
The insect pathogenic virus can be used either as bio-pesticides in prevention and control of pests, or be applied in studies of pathogenic genes and viral expression vectors. The priority is to have enough sources of the insect pathogenic virus. The insect-infecting baculovirus needs to be cultured in live cells, such as propagation through insect larvae or cell line culture. Among them, multiplication of MaviMNPV involved the large-scale rearing of M. vitrata larvae. This process is labor intensive and several challenges need to be met (e.g. the conditions for breaking embryonic diapause, the development of artificial diets, and the prevention of epizootic diseases). In addition, M. vitrata larvae are quite small; the yields of virus are thus limited. In vitro production of MaviMNPV in a highly susceptible insect cell line is an alternative solution. This strategy possesses advantages include no contamination from other microorganisms during cultivation, screening, and maintaining highly virulent MaviMNPV strains.
Insect cell lines have the advantage for in vitro culture of virus or pathogenic microorganisms. Hundreds of continuous cell lines have been established from over 100 insect species since Grace et al. established the first insect cell line in 1962 (Lynn, D. E., Development of insect cell lines: virus susceptibility and applicability to prawn cell culture. Methods in Cell Sci., 21(4):173-81.1999). These cell lines have been used broadly in researches of physiology, histology, embryology, molecular biology, pathology and insect virology. The baculovirus expression vectors and the production of recombinant proteins also need insect cell lines. The advantages of recombinant proteins produced by baculovirus include high-level expression, high bio-activity, low cost, and easy manipulation. However, there is still no MaviMNPV permissive cell line established. Besides, virus has specificity to host cell, each cell has different susceptibility to different virus. Therefore, establishment of a NPV permissive insect cell line is critical for basic research and the following application.
In summary, the production of biopesticides can be enhanced to reach the safety and pest control purposes if a highly susceptible cell line to MaviMNPV or other insect pathogenic microorganisms is established. The cell line can also be applied in the expression system of insect baculovirus to produce recombinant proteins for medical or research purposes.